Low frequency changes on the surfaces of substrates are possible indications of contamination and/or abnormalities. Examples include lead in painted surfaces; scaling inside pipes; mold on carpeting, walls, or heating and cooling ducts; bacteria on skin; and cancer cell receptors. Without early detection and subsequent remediation, these surface modifications can lead to deleterious health, economic, environmental, and structural damage. An area wherein surface modification detection is particularly needed involves the early diagnosis of certain cancers.
A serious global health issue concerns oral cancer and its lack of early detection. Head and neck squamous cell carcinoma (HNSCC) makes up about 4% of all newly diagnosed cancers in the U.S. (30,000 new cases per year), with secondary primary tumors developing at a rate of 2-3% new cases per year (Tabor et al., “Multiple Head and Neck Tumors Frequently Originate from a Single Preneoplastic Lesion” Amer. J. Path., 161 (3) 1051-1060 (2002). In fact, long term survival rates have not improved significantly in the last 20 years. A major preventable cause is thought to be tobacco use; especially the use of chewing tobacco. In some Asian countries, this tobacco use is combined with chewing of betal leaves, and oral cancer rises to 40% of new cancers (Pande et al., “Prognostic Factors in Betel and Tobacco Related Oral Cancer”, Oral Onocology, 38, 491-499 (2002).
Early detection is hampered because pain and other symptoms are normally not present during early stages. Accordingly, diagnosis usually occurs at a late stage, such as after the primary tumor has metastasized and presented with a lump in the neck. Such advanced cases have a very bad prognosis that is worse than that of breast or prostate cancer (Canto, M. et al., “Views of Oral Cancer Prevention and Early Detection: Maryland Physicians,” Oral Onc. 38, 373-377 (2002)).
In addition, successful treatment of HNSCC frequently involves radiation therapy and leaves the patient with significant deficits in quality of life factors including decreased saliva production. Surgical removal of deeper lesions, which result from invasion initiated at the surface, also can be very traumatic (e.g., removal of large parts of the tongue or jaw). Early detection allows less invasive treatment, a more positive effect on survival, and a decreased risk of physical dysfunction. Unfortunately, no effective and easily administered screening test is currently available.
Currently, early detection is difficult since early lesions are small, requiring a careful physical examination to detect. For instance, careful examination requires grasping the tongue with gauze, distracting it to a contralateral position, and withdrawing it as far as possible from the oral cavity. Lighting is difficult, since head mirrors, lamps and penlights lack sufficient intensity and color balance to detect the most common pre-cancerous lesions (Mashberg, S. et al., “Early Diagnosis of Asymptomatic Oral and Oropharyngeal Squamous Cancers,” CA Cancer J. Clin. 45, 328-51 (1995)). A recent survey of Maryland physicians found that 82% did not routinely do an oral examination, and many felt (incorrectly) that the disease was rare. Some expressed surprise that it was more common than cervical cancer, where screening was routinely done, but expressed the view that a continuing education class in detecting oral cancer would draw little interest (Canto, M. et al., “Views of Oral Cancer Prevention and Early Detection: Maryland Physicians,” Oral Onc. 38, 373-377 (2002). An easy to use, accurate screening test to detect oral cancers in the early onset of the disease would be highly beneficial.
Toluidine blue dye has been used to aid in the detection of oral cancer because it is a vital stain that colors various lesions for easier visibility during oral examinations (Mashberg, S. et al. “Early Diagnosis of Asymptomatic Oral and Oropharyngeal Squamous Cancers,” CA Cancer J. Clin. 45, 328-51 (1995)). However, toluidine also stains non-malignant lesions, such as ulcers, requiring additional examination after a period of time sufficient for healing to take place. In addition, the use of toluidine to stain lesions requires direct observation by a health care professional.
Certain molecules are known as indicators for cancerous growths. For example, cell surface receptors for the vitamin folic acid are overexpressed on most human cancer cells, including those of the mucosa (Wang S, Low P S., “Folate-Mediated Targeting of Antineoplastic Drugs, Imaging Agents, and Nucleic Acids to Cancer Cells” J. Control Release 1998 Apr. 30, 53 (1-3): 39-48) and other epithelial malignancies of the head and neck (Lu, Y and Low, P., “Folate-Mediated Delivery of Macromolecular Anticancer Therapeutic Agents,” Advanced Drug Delivery Reviews 54, 675-693 (2002). Additional surface receptors with known overexpression in HNSCC are those for transferrin (TF), epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF).
The folic acid molecule has three fragments chemically linked: 6-methylpteridin, p-amino benzoic acid (PABA), and glutamic acid. Naturally occurring folic acid can have up to eight glutamic acid residues linked together through the carboxylate group on the amino acid's side chain.
Folate is needed in the synthesis of DNA nucleotides and the methyl group of thymine. Hence, folate is required in rapidly growing cell populations, and more rapid uptake is required since it cannot be synthesized by the cell. Syntheses of cell-surface receptors for the molecule are upregulated, and the receptors mediate both membrane transport and endocytosis of the free folate and folate conjugates.
Because of this enhanced level of folate receptor on the cell surface, attachment of folate to genes, contrast agents, liposomes, drugs, dyes and other markers has been frequently used to specifically target cancer cells in the presence of large amounts of normal tissue (Wang S, Low P S., “Folate-Mediated Targeting of Antineoplastic Drugs, Imaging Agents, and Nucleic Acids to Cancer Cells” J. Control Release 1998 Apr. 30, 53 (1-3): 39-48); Lu, Y and Low, P., “Folate-Mediated Delivery of Macromolecular Anticancer Therapeutic Agents,” Advanced Drug Delivery Reviews 54, 675-693 (2002); Reddy J A, Dean D, Kennedy M D, Low P S “Optimization of Folate-Conjugated Liposomal Vectors for Folate Receptor-Mediated Gene Therapy” J. Pharm Sci., 1999 November; 88 (11): 1112-8; Lu, Y and Low, P., “Folate-Mediated Delivery of Macromolecular Anticancer Therapeutic Agents,” Advanced Drug Delivery Reviews 54, 675-693 (2002; Das C R, Su T. “Particle-Mediated Intravascular Delivery of Oligonucleotides to Tumors: Associated Biology and Lessons from Genotherapy” Drug Deliv. 8 (4): 191-213, 2001). When using folate as a conjugated targeting agent, it was found that the site of folate attachment had to be at the γ-carboxylate group to allow bonding of the other end to the receptors (Leamon, C and Low, P., “Delivery of Macromolecules into Living Cells: A Method that Exploits Folate Receptor Endocytosis” Proc. Natl. Acad. Sci USA 88, 5572-5576 (1991). There are at least two types of cell surface receptors for folate (α and β), which both have very high binding affinities (nano molar) and vary in levels with cell type (Kranz D M, Patrick T A, Brigle K E, Spinella M J, Roy E J “Conjugates of Folate and Anti-T-Cell-Receptor Antibodies Specifically Target Folate-Receptor-Positive Tumor Cells for Lysis” Proc Natl Acad Sci USA 1995 Sep. 26; 92 (20): 9057-61).
One carrier mechanism for delivering substances to various locations is the microsphere. Microspheres containing surface modifications can be manufactured that are both pH-sensitive and magnetic. Many good methods exist to make microspheres in different size ranges from a wide range of polymers that incorporate useful components such as dyes, drugs or magnetic powders (Karsa, D. and Stephenson, R., “Encapsulation and Controlled Release” The Royal Society of Chemistry, Pulisher, Cambridge, UK. 1993; Hafeli, U. et al., ed., “Scientific and Clinical Applications of Magnetic Carriers” Plenum Press, NY (1997); Batich, C., Jun, Y., Bucaria, C and Elsabee, M. “Swelling Behavior of pH-Sensitive Copolymers Based on Styrene and 4- (or 2-) Vinylpyridine” Macromolecules, 126, pp. 4675-4681 (1993). Surface modification of microspheres for targeting purposes is also a well-established technology, and many types of these particles are available commercially from suppliers such as Bangs Laboratories. Methods of immobilizing folate, in particular, have been developed using di-functional coupling agents (Dauty et al. 2002 July-August; 13 (4): 831-9 Intracellular delivery of nanometric DNA particles via the folate receptor).
Diagnostic applications utilizing microspheres are known (U.S. Pat. Nos. 6,268,222; 6,327,410; 6,417,340 and 6,495,325). Some use color changes as indications for diagnosis; others do not.
U.S. Pat. No. 6,221,579 to Everhart et al., describes a diagnostic system using functionalized microspheres that bind with analytes. The technology of the '579 patent is specifically engineered for in vitro use. A polymer film is purposely adapted to allow light diffraction when the microspheres are bound to the desired analyte.
U.S. Pat. No. 5,919,633 to Tausk et al., describes a diagnostic method utilizing liposomes that change color in the presence of a target antigen. The surface of the liposomes are coated with an antigenic structures that break down the liposome in the presence of the target antigen releasing the color changing marker. The '633 patent pertains to testing taking place outside the body. The diagnostic marker is in the change of color, not in the degree of change.
It is an object of the present invention to provide a method to detect a wide variety of changes on surfaces.
To fill the current void in early diagnostic methods in detecting cancer cells, it is an object of the present invention to provide a method for detecting cancer cells in vivo. It is a further object to provide a method for diagnosing HNSCC in oral mucosa. It is also an object to provide a kit for home or clinical use for diagnosing HNSCC.